Journal: bioRxiv

Article Title: Specific SOX10 enhancer elements modulate phenotype plasticity and drug resistance in melanoma

doi: 10.1101/2024.12.12.628224

Figure Lengend Snippet: Schematic illustrating SOX10 dependency in human melanoma cell lines based on Chronos dependency scores. WM115 cells exhibit a high dependency on SOX10, while A375 cells show only moderate dependency. 2b. Generation of enhancer deletion lines. CRISPR/Cas9 and guide RNAs targeting enhancer elements were electroporated into both cell lines, followed by single cell sorting and genotyping to confirm successful deletion. Tolerance of WM115 and A375 of the targeted deletion of enhancer elements. 2b (top): The highly SOX10 dependent WM115 cells showed low tolerance for enhancer deletion, with only ∼13% of clones harboring the deletion surviving. 2b (bottom): The less SOX10-dependent A375 cells demonstrated higher tolerance to enhancer deletion, with ∼80% of the deletion clones surviving. 11 stable deletion lines were generated: each line is labeled as follows: ‘Parental Cell Line’_‘Region Number’. ‘Replicate Number’. Additionally, wild type (WT) A375 and WM115 cells underwent the same process of single-cell sorting and clonal expansion to serve as controls, ensuring consistency in selection conditions.

Article Snippet: WM115 cells were purchased from Fisher Scientific (NC1926427) and were maintained in Tu2% medium, prepared as follows: 1 L of MCDB medium was prepared by dissolving 1 bottle of MCDB (cat # M74031L) in 1 L of ddH20 with 1.2 g of sodium bicarbonate.

Techniques: CRISPR, FACS, Clone Assay, Generated, Labeling, Selection

Journal: bioRxiv

Article Title: Specific SOX10 enhancer elements modulate phenotype plasticity and drug resistance in melanoma

doi: 10.1101/2024.12.12.628224

Figure Lengend Snippet: Normalized SOX10 mRNA expression levels measured by qPCR in A375 cells with and without SOX10 enhancer deletions (A_WT, A_1.1, A_1.2, A_4.1, A_4.2). Deletion of enhancer elements resulted in significant reductions in SOX10 expression in several lines, with varying magnitudes. Statistical significance: *p < 0.05, **p < 0.01. (Center) Proliferation curves of A375 cell lines measured by CellTiter-Glo luminescence assay over 7 days. Deletion lines showed slightly reduced proliferation compared to the A_WT control. Statistical significance across time points: ****p < 0.0001. (Right) Principal Component Analysis (PCA) of A375 deletion line RNA-seq data, colored by k-means clustering (k=3). Clustering reflects subtle shifts in gene expression profiles across deletion lines, silhouette width cluster optimization. 3b. WM115 Cell Lines (Left) Normalized SOX10 mRNA expression levels measured by qPCR in WM115 cells with and without SOX10 enhancer deletions. Deletion of enhancer elements significantly reduced SOX10 expression in most lines. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant. (Center) Proliferation curves of WM115 cell lines measured by CellTiter-Glo luminescence assay over 7 days. Deletion lines exhibited significantly increased proliferation relative to W_WT. Statistical significance across time points: ****p < 0.0001. (Right) PCA of WM115 deletion line RNA-seq data, colored by k-means clustering (k=4). Clustering highlights distinct shifts in transcriptional profiles, reflecting differentiation state transitions upon enhancer deletion, silhouette width cluster optimization.

Article Snippet: WM115 cells were purchased from Fisher Scientific (NC1926427) and were maintained in Tu2% medium, prepared as follows: 1 L of MCDB medium was prepared by dissolving 1 bottle of MCDB (cat # M74031L) in 1 L of ddH20 with 1.2 g of sodium bicarbonate.

Techniques: Expressing, Luminescence Assay, Control, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Specific SOX10 enhancer elements modulate phenotype plasticity and drug resistance in melanoma

doi: 10.1101/2024.12.12.628224

Figure Lengend Snippet: . Phenotype trajectory scores across deletion lines: Mean phenotype trajectory scores and statistical comparisons (Dunnett’s test) for A375 and WM115 cell lines with and without SOX10 enhancer deletions, based on weighted expression of Tsoi sub-phenotype gene lists (melanocytic to undifferentiated, 1–7). Scores for WM115 deletion lines (W_1.2, W_4.1, W_4.2) show significant shifts towards more undifferentiated phenotypes compared to W_WT. In A375, only A_1.2 exhibits a statistically significant shift, though trends are evident in A_4.1 and A_4.2. Arrow plot (right) visualizes the magnitude and direction of shifts in phenotype scores relative to WT parental lines. . Boxplots of weighted phenotype scores: Weighted phenotype trajectory scores for A375 (top) and WM115 (bottom) deletion lines. WM115 lines exhibit more pronounced and statistically significant shifts towards undifferentiated states compared to A375 lines. Adjusted p-values: **p < 0.01, ***p < 0.001, ns: not significant. . Heatmap of phenotype category scores: Heatmap of individual sub-phenotype category scores (adapted from Tsoi et al. gene lists) for each deletion line.

Article Snippet: WM115 cells were purchased from Fisher Scientific (NC1926427) and were maintained in Tu2% medium, prepared as follows: 1 L of MCDB medium was prepared by dissolving 1 bottle of MCDB (cat # M74031L) in 1 L of ddH20 with 1.2 g of sodium bicarbonate.

Techniques: Expressing

Journal: bioRxiv

Article Title: Specific SOX10 enhancer elements modulate phenotype plasticity and drug resistance in melanoma

doi: 10.1101/2024.12.12.628224

Figure Lengend Snippet: Cell viability of A375 cells challenged with (left): dabrafenib and (right): trametinib. All lines show an increased IC50, indicating greater drug resistance compared to their WT counterparts. (5b) Cell viability of WM115 cells challenged with (left): dabrafenib and (right): trametinib. Similar to A375, almost all deletion lines exhibit increased IC50 values, suggesting a higher level of drug resistance than the WT. (5c) Bar graphs showing the IC50 values for each line under both drug conditions. IC50 values for each deletion line are plotted for dabrafenib (left), and trametinib (right), highlighting the differences in drug resistance across the various lines.

Article Snippet: WM115 cells were purchased from Fisher Scientific (NC1926427) and were maintained in Tu2% medium, prepared as follows: 1 L of MCDB medium was prepared by dissolving 1 bottle of MCDB (cat # M74031L) in 1 L of ddH20 with 1.2 g of sodium bicarbonate.

Techniques:

Journal: Cancer research

Article Title: PTEN Lipid Phosphatase Activity Suppresses Melanoma Formation by Opposing an AKT/mTOR/FRA1 Signaling Axis

doi: 10.1158/0008-5472.CAN-23-1730

Figure Lengend Snippet: A, Proliferation as measured by relative confluence of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. B, Quantification of percentage occupied area of low-density colony formation assays of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. C, Quantification of colony number of anchorage-independent growth of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. D, Quantification of cell numbers of transwell invasion assays of M10M6 and M10M1 cells expressing Pten WT or Pten C124S or Pten WT +Fra1. E-F, Quantification of cell numbers of transwell invasion assays of FRA1 silenced mouse melanoma cells M10M6 (E) and human melanoma cells WM115, 1205Lu, and WM2664 (F). G-H, M10M6 cells expressing Pten WT or Pten C124S or Pten WT +Fra1 were subcutaneously injected into NSG mice (n=10). Mice were fed chow containing 200 mg/kg Doxycycline to induce expression of PTEN mutants. Tumor volumes were measured every 3 days. The curves of tumor volume (G) and the tumor weight at the end point (H) are shown. Mean ± SEM are shown in (A-H). Data are analyzed with Student’s unpaired t test, * P<0.05, ** P<0.01, *** P<0.001.

Article Snippet: Human melanoma cell lines WM115 (RRID:CVCL_0040), WM266–4 (RRID:CVCL_2765), and 1205Lu (RRID:CVCL_5239) were obtained from Meenhard Herlyn (Wistar Institute) and SBCL2 (RRID:CVCL_D732) were obtained from David Tuveson (Cold Spring Harbor Laboratory).

Techniques: Over Expression, Expressing, Injection